PURIFYING HOMOGENEOUS SPERMATID SUB-POPULATIONS

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DESCRIPTION (Adapted from applicant's description): Spermiogenesis is a sequential assembly process based on carefully timed translation of stored mRNAs in transcriptionally silent cells; however, the molecular mechanisms that determine when individual mRNAs will be translated are not well understood. A large part of the problem results from our having no reliable method of purifying sub-populations of spermatids at different stages of maturation for molecular analysis. The proposed study aims to resolve this deficiency. The applicant proposes to develop a method that uses transgenic mice that expresses the jellyfish green fluorescent protein (GFP) in post- meiotic male germ cells, in combination with fluorescence-activated cell sorting (FACS), to purify homogeneous sub-populations of staged spermatids. The goals of this project are: 1) Isolate at least 20 spermatid sub- populations by FACS. 2) Evaluate the purity and stage of spermatids represented in each sub-population based on morphological, ultrastructural, and molecular criteria; select 10 homogeneous sub-populations representing different stages spanning all of spermiogenesis. 3) Purify mRNP particle- associated mRNA (stored information) and polysome-associated mRNA (active information) from each of these homogeneous sub-populations, prepare a cDNA library from each mRNA sample, and evaluate each by RT-PCR. This work will establish a reliable protocol for sorting spermatids and will yield ordered libraries of most or all of the active and stored spermiogenic information. Our long-term goals are to use this technology to catalog and characterize most or all spermiogenic mRNAs and to determine when each one is used during sperm maturation. In the current submission, the applicant is requesting two years of R03 funding to rigorously establish the technology on which this research, as well as countless other molecular investigations on spermiogenesis, can be founded.